Method and apparatus for detection of rare cells

ABSTRACT

Fluorescent dye-tagged rare cells are disposed in a biological fluid layer contained in an annular gap ( 12 ) between an inside test tube wall ( 14 ) and a float wall ( 16 ). One or more analysis images are acquired at one or more focus depths using a microscope system ( 10, 10′, 10″, 10 ′″). Each acquired analysis image is image processed each to identify candidate rare cell images. The test tube ( 72 ) is selectively rotated. The test tube and the microscope system are selectively relatively translated along a test tube axis ( 75 ). The acquiring and image processing are repeated for a plurality of fields of view accessed by the selective rotating and selective relative translating and substantially spanning the annular gap.

The present application is a divisional application of U.S. patentapplication Ser. No. 11/261,105, filed Oct. 27, 2005.

This application claims the benefit of U.S. Provisional Application No.60/631,025, filed Nov. 24, 2004, which is incorporated by referenceherein in its entirety. This application also claims the benefit of U.S.Provisional Application No. 60/631,026, filed Nov. 24, 2004, which isincorporated by reference herein in its entirety. This application alsoclaims the benefit of U.S. Provisional Application No. 60/631,027, filedNov. 24, 2004, which is incorporated by reference herein in itsentirety.

BACKGROUND

The following relates to the biological assay arts. It is described withparticular reference to example embodiments that relate to detection ofrare cells, such as epithelial cells, in the buffy coat of a centrifugedblood sample. However, the following relates more generally toidentifying fluorescent dye-tagged cells in a biological fluid layerusing a fluorescent imager, in which there is less than about onedye-tagged cell per optical field-of-view of the fluorescent imager.

In the technique of quantitative buffy coat analysis, a whole bloodsample is drawn and processed using anti-coagulant additives,centrifuging, and so forth to separate the blood into componentsincluding a buffy coat component comprised principally of white bloodcells. Rare cells of interest which are present in the buffy coat, suchas certain epethilial cells associated with certain cancers, are taggedusing a suitable fluorescent dye, and fluorescent imaging is then usedto count the fluorescent dye-tagged cells of interest. Such quantitativebuffy coat analysis is a promising non-invasive technique for screeningfor certain pathologies. For example, some cancer screening techniqueshave been proposed which are based on quantitative buffy coat analysis.

The concentration of rare fluorescent dye-tagged cells in the buffy coatis low—a typical fluorescent imager optical field-of-view may notcontain any fluorescent dye-tagged cells at all. This makes it difficultto focus the fluorescent imager. Additionally, typical substrates orsample holders for optical viewing, such as glass slides or test tubes,have surface height variations substantially exceeding the depth-of-viewof typical fluorescent images. Thus, as the field-of-view of thefluorescent imager is scanned the buffy layer sample may go into and outof focus, and the optical imager should be frequently refocused.Moreover, depending upon how the buffy coat layer sample is mounted, itmay be thicker than the depth-of-view of the fluorescent imager.Fluorescent dyed-cells that are outside of the depth of view will notappear as sharp, well-defined features in the fluorescent images. Inview of these difficulties, locating fluorescent dye-tagged cells in thefluorescent images is challenging.

INCORPORATION BY REFERENCE

U.S. application Ser. No. 10/263,974 filed Oct. 3, 2002 and published asU.S. Publ. Appl. No. 2004/0067162 A1 on Apr. 8, 2004, is incorporated byreference herein in its entirety.

U.S. application Ser. No. 10/263,975 filed Oct. 3, 2002 and published asU.S. Publ. Appl. No. 2004/0067536 A1 on Apr. 8, 2004, is incorporated byreference herein in its entirety.

U.S. patent application Ser. No. ______ filed concurrently with thepresent application, entitled “Optical System for Cell Imaging”,inventor John S. Laudo, and corresponding to attorney docket no. BATZ 200008, is incorporated by reference herein in its entirety.

U.S. patent application Ser. No. ______ filed concurrently with thepresent application, entitled “Sample Tube Handling Apparatus”,inventors Steve Grimes, Thomas D. Haubert, and Eric R. Navin, andcorresponding to attorney docket no. BATZ 2 00010, is incorporated byreference herein in its entirety.

BRIEF SUMMARY

According to one aspect, a method is disclosed for identifyingfluorescent dye tagged rare cells in a biological fluid layer containedin an annular gap between an inside test tube wall and a float wall. Oneor more analysis images are acquired at one or more focus depths using amicroscope system. Each acquired analysis image is image processed eachto identify candidate rare cell images. The test tube is selectivelyrotated. The test tube and the microscope system are selectivelyrelatively translated along a test tube axis. The acquiring and imageprocessing are repeated for a plurality of fields of view accessed bythe selective rotating and selective relative translating andsubstantially spanning the annular gap.

According to another aspect, an apparatus is disclosed comprising anon-volatile storage medium encoding instructions executable by adigital processor to perform a method for identifying fluorescentdye-tagged cells in microscope-acquired analysis images of a biologicalfluid layer having a density of the dye-tagged cells less than about onedye-tagged cell per analysis image. The method comprises imageprocessing each analysis image to identify candidate features having afeature size substantially corresponding to an expected size offluorescent dye tagged cell images in the analysis images.

According to another aspect, a method is disclosed for identifyingfluorescent dye-tagged cells in a biological fluid layer using amicroscope system having a field of view at a selectable focus depth.The microscope field of view is substantially smaller than an area ofthe biological fluid layer. A depth of the biological fluid layer isidentified based on a background fluorescence of the biological fluidlayer caused by the fluorescent dye tagging. One or more analysis imagesare acquired using the microscope system at one or more focus depths ator close to the identified depth of the biological fluid layer. Theacquired one or more analysis images are image processed to identifycandidate features that may correspond to dye tagged cells. The field ofview and the biological fluid layer are relatively moved. Theidentifying, acquiring, image processing, and relative moving arerepeated to identify candidate features over the area of the biologicalfluid layer.

Numerous advantages and benefits of the present invention will becomeapparent to those of ordinary skill in the art upon reading thefollowing detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may take form in various components and arrangements ofcomponents, and in various process operations and arrangements ofprocess operations. The drawings are only for the purpose ofillustrating preferred embodiments and are not to be construed aslimiting the invention.

FIG. 1 diagrammatically shows a microscope system including an opticalsystem for providing substantially uniform static illumination oversubstantially the entire microscope field of view.

FIG. 2 diagrammatically shows the microscope system of FIG. 1 with amodified optical system.

FIG. 3 diagrammatically shows the microscope system of FIG. 1 withanother modified optical system.

FIG. 4 diagrammatically shows the microscope system of FIG. 1 with yetanother modified optical system.

FIGS. 5-10 show various views of a test tube holder:

FIG. 5 shows a perspective view of the holder, with the housing shown inphantom to reveal internal components.

FIG. 6 shows a side view of the holder, with the housing shown inphantom.

-   -   FIG. 7 shows a perspective view of the test tube and alignment        and biasing bearings.

FIG. 8 shows a top view of the test tube holder including an indicationof bias force.

FIG. 9 shows a side view of a second end of the test tube including acontoured base.

FIG. 10 shows a top view of the rotational coupler including a contourconfigured to mate with the contoured base of the test tube shown inFIG. 9.

FIGS. 11A and 11B show top views of another embodiment test tube holder,with a test tube having an eccentric cross-section loaded.

FIG. 12 shows a side view of a portion of a test tube holder employingtilted roller bearings.

FIG. 13 shows a side view of a portion of a test tube holder employingtilted roller bearings staggered along a test tube axis, along with afloat having helical ridges enables spiral scanning of the test tube.

FIG. 14 shows a perspective view of a test tube holder that holds thetest tube horizontally and uses the test tube as a bias force.

FIG. 15 shows a top view of a test tube holder employing bushingsurfaces as alignment bearings and a set of ball bearings as biasbearings.

FIG. 16 diagrammatically depicts certain measurement parameters relevantin performing quantitative buffy coat analysis using a buffy coat sampletrapped in an annular gap between an inside test tube wall and an outersurface of a float.

FIG. 17 diagrammatically shows a suitable quantitative buffy coatmeasurement/analysis approach.

FIG. 18 diagrammatically shows another suitable quantitative buffy coatmeasurement/analysis approach.

FIG. 19 diagrammatically shows a suitable image processing approach fortagging candidate cells.

FIG. 20 shows a pixel layout for a square filter kernel suitable for usein the matched filtering.

FIG. 21 shows a pixel intensity section A-A of the square filter kernelof FIG. 20.

FIG. 22 diagrammatically shows a suitable user verification process forenabling a human analyst to confirm or reject candidate cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

With reference to FIG. 1, a microscope system 10 images a microscopefield of view coinciding with a buffy coat sample disposed in agenerally planar portion of an annular gap 12 between alight-transmissive test tube wall 14 and a float wall 16 of a floatdisposed in the test tube. Suitable methods and apparatuses foracquiring and preparing such buffy coat samples are disclosed, forexample, in U.S. Publ. Appl. No. 2004/0067162 A1 and U.S. Publ. Appl.No. 2004/0067536 A1.

The microscope field of view is generally planar in spite of thecurvatures of the test tube and the float, because the microscope fieldof view is typically much smaller in size than the radii of curvature ofthe test tube wall 14 and the float wall 16. Although the field of viewis substantially planar, the buffy coat sample disposed between thelight-transmissive test tube wall 14 and the float wall 16 may have athickness that is substantially greater than the depth of view of themicroscope system 10.

The test tube is mounted in fixed position respective to the microscopesystem 10 in a manner conducive to scanning the microscope field of viewacross the annular gap. As will be discussed, suitable mechanisms arepreferably provided for effectuating relative rotational and/ortranslational scanning of the field of view over the annular gapcontaining the buffy coat sample.

The microscope system 10 includes a laser 18, such as a gas laser, asolid state laser, a semiconductor laser diode, or so forth, thatgenerates source light 20 (diagrammatically indicated in FIG. 1 bydashed lines) in the form of a laser beam having an illuminationwavelength and a non-uniform spatial distribution that is typicallyGaussian or approximately Gaussian in shape with a highest intensity ina central region of the beam and reduced intensity with increasingdistance from the beam center. An optical train 22 is configured toreceive the spatially non-uniform source light 20 and to output acorrected spatial distribution.

A beam spreader includes a concave lens 24 that generally diverges thelaser beam, and a collimating lens 26 that collimates the spread beam ata larger diameter that substantially matches the diameter of a Gaussianspatial characteristic of a beam homogenizer 30. The beam homogenizer 30flattens the expanded laser beam by substantially homogenizing theGaussian or other non-uniform distribution of the source light toproduce output light having improved spatial uniformity.

In some embodiments, the beam homogenizer 30 operates by having aspatially non-uniform absorption profile that corresponds to aninverse-Gaussian. In such embodiments, the beam homogenizer has highestabsorption in a central region corresponding to the highest intensitycentral region of the expanded laser beam, and has a lower absorption,or no absorption, in the periphery corresponding to the lower intensityouter regions of the expanded laser beam.

In other embodiments, the beam homogenizer 30 refractively redistributesthe light to homogenize the light intensity across the area of theexpanded laser beam, for example using a suitable lens pair. Refractivebeam homogenizers refract light from the high intensity central regionof the expanded laser beam into the lower intensity periphery regions.

A focusing lens 34 and cooperating lenses 36 reduce the expanded andflattened or homogenized laser beam down to a desired beam diameter forinput to an objective 40 that is focused on the microscope field ofview. A dichroic mirror 44 is selected to substantially reflect light atthe wavelength or wavelength range of the laser beam, and tosubstantially transmit light at the fluorescence wavelength orwavelength range of the fluorescent dye used to tag rare cells in thebuffy coat sample.

The optical train 22 including the stationary optical components 24, 26,30, 34, 36 is configured to output a corrected spatial distribution tothe objective 40 that when focused by the objective 40 at the microscopefield of view provides substantially uniform static illumination oversubstantially the entire microscope field of view. The objective 40focuses the corrected illumination onto the microscope field of view.The objective 40 may include a single objective lens, or may include twoor more objective lenses. The focus depth of the microscope system 10 isadjustable, for example by adjusting a distance between the objective 40and the light-transmissive test tube wall 14. Additionally oralternatively, the focus depth may be adjusted by relatively moving twoor more lenses or lensing elements within the objective 40.

The beam homogenizer 30 is designed to output a substantially uniformhomogenized beam for a Gaussian input beam of the correct diameter.However, the objective 40 typically introduces some spatialnon-uniformity. Accordingly, one or more of the stationary opticalcomponents, such as the spreading lens 24, collimating lens 26, focusinglens 34, and/or focusing lenses 36 are optionally configured tointroduce spatial non-uniformity into the spatial distribution such thatthe beam when focused by the objective 40 provides substantially uniformstatic illumination of the microscope field of view. In somecontemplated embodiments, this corrective spatial non-uniformity isintroduced by one or more dedicated optical components (not shown) thatare included in the optical train 22 for that purpose.

The substantially uniform static illumination of the microscope field ofview causes fluorescence of any fluorescent dye-tagged epithelial cellsdisposed within the microscope field of view. Additionally, thefluorescent dye typically imparts a lower-intensity backgroundfluorescence to the buffy coat. The fluorescence is captured by theobjective 40, and the captured fluorescence 50 (diagrammaticallyindicated in FIG. 1 by dotted lines) passes through the dichroic mirror44, and through an optional filter 52 for removing any stray sourcelight, to be imaged by a camera system 56. The camera system 56 may, forexample, include a charge coupled device (CCD) camera for acquiringelectronic images that can be stored in a computer, memory card, orother non-volatile memory for subsequent image processing.

With reference to FIGS. 2 and 3, other suitable microscope systems aredescribed.

FIG. 2 shows a microscope system 10′ that is similar to the microscopesystem 10 of FIG. 1, except that the optical train 22′ differs in thatthe stationary beam homogenizer 30 of FIG. 1 is replaced by a stationarydiffuser 30′. The diffuser 30′ may, for example, be a holographicdiffuser available, for example, from Physical Optics Corporation(Torrance, Calif.). Such holographic diffusers employ a hologramproviding randomizing non-periodic optical structures that diffuse thelight to impart improved spatial uniformity. However, the diffusion ofthe light also imparts some concomitant beam divergence. Typically,stronger diffusion of the light tend to impart more spatial uniformity,but also tends to produce greater beam divergence. Holographic diffusersare suitably classified according to the full-width-at-half-maximum(FWHM) of the divergence angle, with larger divergence angles typicallyproviding more diffusion and greater light uniformity, but also leadingto increased light loss in the microscope system 10′ due to increasedbeam divergence.

In some embodiments of the microscope system 10′, the diffuser 30′ is alow-angle diffuser having a FWHM less than or about 10°. Lower anglediffusers are generally preferred to provide less divergence and hencebetter illumination throughput efficiency; however, if the divergenceFWHM is too low, the diffuser will not provide enough light diffusion toimpart adequate beam uniformity. Low diffusion reduces the ability ofthe diffuser 30′ to homogenize the Gaussian distribution, and alsoreduces the ability of the diffuser 30′ to remove speckle.

With reference to FIG. 3, another embodiment microscope system 10″ issimilar to the microscope system 10′, and includes an optical train 22″that employs a diffuser 30″ similar to the diffuser 30′ of themicroscope system 10′. However, the diffuser 30″ is tilted at an angle θrespective to the optical path of the optical train 22″ so as tosubstantially reduce a speckle pattern of the source light 20. Withoutbeing limited to any particular theory of operation, it is believed thatthe tilting shifts the speckle pattern to higher spatial frequencies, ineffect making the speckle size smaller. The speckle size is spatiallyshifted by the tilting such that the frequency-shifted speckle issubstantially smaller than an imaging pixel size.

In some embodiments, a tilt angle θ of at least about 30° respective tothe optical path of the optical train 22″ is employed, which has beenfound to substantially reduce speckle for diffusers 30″ having a FWHM aslow as about 5°. On the other hand, tilt angles θ of greater than about45° have been found to reduce illumination throughput efficiency due toincreased scattering, even for a low-angle diffuser having a FWHM of 5°.

With reference to FIG. 4, it is to be appreciated that the microscopesystems disclosed herein are suitable for other microscopy applicationsbesides imaging of samples contained in or supported by test tubes. InFIG. 4, a microscope system 10′″ includes a light emitting diode (LED)18′″ as the light source, rather than the laser 18 used in the previousmicroscope systems 10, 10′, 10″. Because the LED 18′″ outputs divergingsource light 20′″ rather than a collimated laser beam, an optical train22′″ is modified in that the beam-expanding concave lens 24 is suitablyomitted, as shown in FIG. 4. Alternatively, a lens can be included inthe position of the lens 24, but selected to provide a suitabledivergence angle adjustment for collimation by the collimating lens 26.The optical train 10′″ employs a diffuser 30′″ similar to the diffusers30′, 30″. The LED 18′″ outputs incoherent light, and so speckle isgenerally not present. However, the output of the LED 18′″ typicallydoes have a non-Gaussian distribution, for example a Lambertiandistribution. In view of these characteristics of the source light 20′″,the diffuser 30′″ is not tilted, and in some cases the diffuser 30″′ canhave a smaller divergence angle FWHM than the untilted diffuser 30′ usedto impart spatial uniformity to the laser beam source light 20 in themicroscope system 10′ of FIG. 2.

The microscope system 10′″ of FIG. 4 further differs from the microscopesystems 10, 10′, 10″ in that the microscope system 10′″ images a sampledisposed on a planar slide 60, which is optionally covered by anoptional cover glass 62. The slide 60 is disposed on an x-y planartranslation stage 64 to enable scanning across the sample. It will beappreciated that the LED 18′″ and optical train 22′″ are also suitablefor imaging the buffy coat sample disposed in the annular gap 12 betweenthe light-transmissive test tube wall 14 and float wall 16 shown inFIGS. 1-3. Conversely, it will be appreciated that the laser 18 andoptical train 22, 22′, 22″ are also suitable for imaging the planarsample on the slide 60 shown in FIG. 4.

The optical trains 22, 22′, 22″, 22′″ have components which arestationary in the sense that the components are not rotated, relativelyoscillated, or otherwise relatively moved. It is, however, contemplatedto move the optical train and the objective 40 as a whole, and/or toinclude beam-steering elements, or so forth, to enable relative scanningof the field of view respective to the sample.

Suitable microscope systems for imaging an annular sample contained inor supported by a test tube have been described. The annular gap 12typically has a thickness that is substantially larger than a depth ofview of the microscope objective 40. The test tube wall 12 and floatwall 16 are typically not uniform across the entire surface of the testtube or float. While the microscope objective 40 typically has anadjustable depth of focus (adjusted by moving internal opticalcomponents and/or by moving the objective 40 toward or away from thetest tube wall 12), the range of adjustment is limited. Accordingly, thetest tube should be held such that the surface proximate to theobjective 40 is at a well-defined distance away from the objective 40 asthe test tube is rotated and as the objective 40, or the test tube, istranslated along a tube axis.

Suitable test tube holders for achieving such aspects are nextdescribed.

With reference to FIGS. 5-10, a test tube holder 70 has mounted thereina test tube 72 that is sealed by a test tube stopper 73. The sealed testtube 72 contains a float 74 and blood that has been suitably processedand centrifuged to separate out components including red blood cells,plasma, and a buffy coat, for example as described in U.S. Publ.Applications 2004/0067162 A1 and 2004/0067536 A1. The float 74 has adensity which is less than that of the packed red blood cells component(1.090 g/ml) and greater than that of the plasma component (1.028 g/ml).Accordingly, after centrifuging the float 74 is disposed along the testtube axis 75 (drawn and labeled in FIG. 6) between the packed red bloodcell layer and the plasma layer, that is, generally coincident with thebuffy coat. After centrifuging, the buffy coat is generally disposed inthe annular gap 12 between the test tube wall 14 and the float wall 16.(See labeling in FIG. 6). Annular sealing ridges 76, 78 at ends of thefloat 74 engage an inside surface of the test tube 72 when the test tubeis at rest so as to seal the annular gap 12. During centrifuging,however, the test tube 72 expands to provide fluid communication acrossthe ridges 76, 78 so as to enable the buffy coat to substantiallycollect in the annular gap 12.

At least one first alignment bearing, namely two radially spaced apartfirst alignment bearings 80, 81 in the example test tube holder 70, aredisposed on a first side of the annular sampling region 12. At least onesecond alignment bearing, namely two second radially spaced apartalignment bearings 82, 83 in the example test tube holder 70, aredisposed on a second side of the annular sampling region 12 opposite thefirst side of the annular sampling region 12 along the test tube axis75. The alignment bearings 80, 81, 82, 83 are fixed roller bearingsfixed to a housing 84 by fastening members 85 (shown only in FIG. 8).

At least one biasing bearing, namely two biasing bearings 86, 87 in theexample test tube holder 70, are radially spaced apart from thealignment bearings 80, 81, 82, 83 and are spring biased by springs 90 topress the test tube 72 against the alignment bearings 80, 81, 82, 83 soas to align a side of the annular sampling region 12 proximate to theobjective 40 respective to the alignment bearings 80, 81, 82, 83. In theexample test tube holder 70, the two first alignment bearings 80, 81 andthe first biasing bearing 86 are radially spaced apart by 120° intervalsand lie in a first common plane 92 on the first side of the annularsampling region 12. Similarly, the two second alignment bearings 82, 83and the second biasing bearing 87 are radially spaced apart by 120°intervals and lie in a second common plane 94 on the second side of theannular sampling region 12. The springs 90 are anchored to the housing84 and connect with the biasing bearings 86, 87 by members 98.

More generally, the bearings 80, 81, 86 and the bearings 82, 83, 87 mayhave radial spacings other than 120°. For example the biasing bearing 86may be spaced an equal radial angle away from each of the alignmentbearings 80, 81. As a specific example, the biasing bearing 86 may bespaced 135° away from each of the alignment bearings 80, 81, and the twoalignment bearings 80, 81 are in this specific example spaced apart by90°.

Optionally, the first common plane 92 also contains the float ridge 76so that the bearings 80, 81, 86 press against the test tube 72 at theridge 76, and similarly the second common plane 94 optionally alsocontains the float ridge 78 so that the bearings 82, 83, 87 pressagainst the test tube 72 at the ridge 78. This approach reduces alikelihood of distorting the annular sample region 12. The biasingbearings 86, 87 provide a biasing force 96 that biases the test tube 72against the alignment bearings 80, 81, 82, 83.

The housing includes a viewing window 100 that is elongated along thetube axis 75. The objective 40 views the side of the annular sampleregion 12 proximate to the objective 40 through the viewing window 100.In some embodiments, the objective 40 is linearly translatable along thetest tube axis 75 as indicated by translation range double-arrowindicator 104. This can be accomplished, for example, by mounting theobjective 40 and the optical train 22, 22′, 22″, or 22′″ on a commonboard that is translatable respective to the test tube holder 70. Inanother approach, the microscope system 10, 10′, 10″, 10′″ isstationary, and the tube holder 70 including the housing 84 istranslated as a unit to relatively translate the objective 40 across thewindow 100. In yet other embodiments, the objective 40 translates whilethe optical train 22, 22′, 22″, or 22′″ remains stationary, and suitablebeam-steering components (not shown) are provided to input the beam tothe objective 40. The objective 40 is also focusable, for example bymoving the objective 40 toward or away from the test tube 72 over afocusing range 106 (translation range 104 and focusing range 106indicated only in FIG. 6).

Scanning of the annular sampling region 12 calls for both translationalong the test tube axis, and rotation of the test tube 72 about thetest tube axis 75. To achieve rotation, a rotational coupling 110 isconfigured to drive rotation of the test tube 72 about the tube axis 75responsive to a torque selectively applied by a motor 112 connected withthe rotational coupling 110 by a shaft 114. The rotational coupling 110of the example test tube holder 70 connects with the test tube 72 at anend or base thereof. At an opposite end of the test tube 72, aspring-loaded cap 116 presses against the stopper 73 of the test tube 72to prevent the rotation from causing concomitant translational slippageof the test tube 72 along the test tube axis 75.

With particular reference to FIGS. 9 and 10, in some embodiments therotational coupling 110 is a contoured coupling having a contour 120configured to mate with a contoured base 122 of the test tube 72. In theillustrated example of FIGS. 9 and 10, the contour 120 of the coupling110 includes four depressions that receive four nibs of the contouredbase 122 of the test tube 72. Other contour features can be employed.

In some embodiments, the contour 120 and contoured base 122 are keyed bysuitable rotationally asymmetric features 124, 126 (shown in phantom inFIGS. 9 and 10) in the coupling 110 and test tube base 122,respectively, to define an absolute rotational position of the test tube72 when the contoured base 122 of the test tube 72 is mated with thecontour 120 of the rotational coupling 110. In this way, the absoluterotational position (measured, for example as an absolute angle value indegrees) can be maintained even if the test tube 72 is removed from andthen re-installed in the test tube holder 70.

In another approach for providing absolute angular position, the testtube optionally includes fiducial markers, for example opticallyreadable reflective fiducial markers (not shown), to indicate theabsolute rotational position of the test tube.

In some embodiments, the second side alignment roller bearings 82, 83are omitted, and the rotational coupling 110 defines the at least onesecond alignment bearing disposed on the second side of the annularsampling region 12 opposite the first side of the annular samplingregion 12 along the test tube axis 75. In such embodiments, therotational coupling acts as a mechanically driven alignment bearing toprovide both alignment and rotation of the test tube 72. Optionally, insuch embodiments the second side bias bearing 87 is also omitted alongwith the corresponding roller bearings 82, 83.

On the other hand, in some other contemplated embodiments the rotationalcoupling 110 is omitted, and one or more of the roller bearings 81, 82,83, 84, 86, 87 are mechanically driven to rotate the test tube 72. Insuch embodiments, the driven roller bearings serve as the rotationalcoupling. The driven bearings can be one or more of the alignmentbearings 81, 82, 83, 84, or can be one or more of the biasing bearings86, 87.

In order to install the test tube 72 in the test tube holder 70, thehousing 84 is provided with a hinged lid or door 130 (shown open in FIG.5 and closed in FIG. 6). When the hinged lid or door 130 is opened, thespring-loaded cap 116 is lifted off of the stopper 73 of the test tube72. Optionally, the support members 98 that support the biasing bearings86, 87 include a manual handle or lever (not shown) for manually drawingthe biasing bearings 86, 87 away from the test tube 72 against thebiasing force of the springs 90 so as to facilitate loading or unloadingthe test tube 72 from the holder 70.

The test tube holder 70 advantageously can align the illustrated testtube 72 which has straight sides. The test tube holder 70 can alsoaccommodate and align a slightly tapered test tube. The held position ofa tapered test tube is indicated in FIG. 6 by a dashed line 134 whichindicates the tapered edge of a tapered test tube. The illustratedtapering 134 causes the end of the test tube closest to the rotationalcoupling 110 to be smaller diameter than the end of the test tubeclosest to the spring-loaded cap 116. As indicated in FIG. 6, thebiasing of the biasing bearings 86, 87 presses the test tube against thealignment bearings 81, 82, 83, 84 to maintain alignment of the portionof the annular sample region 12 proximate to the objective 40 in spiteof the tapering 134. It will be appreciated that the holder 70 cansimilarly accommodate and align a test tube having an opposite taper inwhich the end closes to the rotational coupling 110 is larger indiameter than the end closest to the spring-loaded cap 116.

In the case of a substantial tapering, or in the case a test tube thathas a highly eccentric or non-circular cross-section, the biasingagainst the alignment bearings 81, 82, 83, 84 will not completelycompensate for the tapering or cross-sectional eccentricity orellipticity. This is because the radial spacing apart of the firstalignment bearings 81, 82 and the radial spacing apart of the secondalignment bearings 83, 84 allows a narrower tube to extend a furtherdistance into the gap between the first alignment bearings 81, 82 andinto the gap between the second alignment bearings 83, 84.

With reference to FIGS. 11A and 11B, a modified test tube 72′ having anelliptical cross-section is more precisely aligned by employing a set ofthree bearings per supported float ridge, in which the three bearingsinclude only one alignment bearing 81′ and two or more biasing bearings86′. The alignment bearing 81′ is at the same radial position as theobjective 40 (shown in phantom in FIGS. 11A and 11B). As the ellipticaltest tube 12′ rotates, the imaged side that is biased against thealignment bearings 81′ remains precisely aligned with the radiallycoincident objective 40 whether the imaged side correspond to the shortaxis of the elliptical test tube 72′ (FIG. 11A), or whether the imagedside correspond to the long axis of the elliptical test tube 72′ (FIG.11B).

With reference to FIG. 12, in another variation, bearings 140 are tiltedrespective to the tube axis 75 of the test tube 72 to impart forcecomponents parallel with the tube axis 75 to push the test tube 72 intothe rotational coupling 110. In this arrangement, the spring-loaded cap116 is optionally omitted, because the tilting of the bearings 140opposes translational slippage of the test tube 72 during rotation.

With reference to FIG. 13, in another variation, a modified float 74′includes spiral ridges 76′, and tilted bearings 142 are spaced along thetube axis 75 in accordance with the spiral pitch to track the spiralingsealing ridges 76′ responsive to rotation of the test tube 72. In thisapproach, the tilted bearings 142 impart a force that causes the testtube 72 to translate along the tube axis 75, so that the objective 40can be maintained at a fixed position without translating while scanningannular gap 12′. In this approach, the roller bearings 142 are suitablymotorized to generate rotation of the test tube 72. That is, the rollerbearings 142 also serve as the rotational coupling.

With reference to FIG. 14, in another variation, the mechanical bias canbe provided by a mechanism other than biasing bearings. In example FIG.14, the test tube 72 is arranged horizontally resting on alignmentbearings 181, 182, 183, 184 with the objective 40 mounted beneath thetest tube 72. A weight 186 of the test tube 72 including the float 74(said weight diagrammatically indicated in FIG. 14 by a downward arrow186) provides as the mechanical bias pressing the test tube 72 againstthe alignment bearings 181, 182, 183, 184. In other contemplatedembodiments, a vacuum chuck, positive air pressure, magnetic attraction,or other mechanical bias is employed to press the test tube against thealignment bearings. The alignment bearings 181, 182, 183, 184 can berotated mechanically so that the alignment bearings 181, 182, 183, 184serve as the rotational coupling, or a separate rotational coupling canbe provided.

With reference to FIG. 15, the bearings can be other than rollerbearings. For example, the bearings can be rollers, ball bearings, orbushing surfaces. In the variant test tube holder shown in FIG. 15, ahousing 200 provides an anchor for a spring 202 that presses a set ofbiasing ball bearings 204 against the test tube 72 to press the testtube 72 against alignment bearings 211, 212 defined by bushing surfacesof the housing 200. Other types of bearings can be used for the biasingand/or alignment bearings that support the test tube as it rotates.

In the illustrated embodiments other than the embodiment of FIG. 13, thetest tube is not translated within the tube holder, and instead thetranslative component of the scanning is achieved by translating theobjective 40, or by translating the test tube and tube holder as a unit.In other contemplated embodiments, it is contemplated to keep theobjective fixed and to translate the test tube within the test tubehousing, for example by including a linear translation capability in theshaft 114 connecting the motor 112 with the rotational coupling 112 soas to translate the test tube 72 along the test tube axis 75.

Suitable microscope systems and test tube holders have been describedfor imaging an annular sample region contained in or supported by a testtube. It is to be understood that the annular sampling region can beother than the illustrated fluid sample contained in the gap 12 betweenthe test tube wall 14 and the float wall 16. For example, the annularsample region can be a film or coating adhered on an outside surface ofthe test tube, or the annular sample region can be a film or coatingadhered on an inside surface of the test tube. Moreover, the term “testtube” is to be broadly construed as encompassing other tubular sampleholders besides the illustrated conventional test tube 72. For example,the test tube could be a cylindrical rod that has been inserted into acontained volume, solid object, or other subject of interest so as tocoat an outside of the cylindrical rod with a sample of the subject ofinterest, or the test tube can be a cylindrical geological core sample,or so forth.

Having described suitable microscope systems and test tube holders foracquiring data from an annular slide or annular sampling regioncontained in or supported by a test tube, suitable processing approachesfor identifying or quantifying fluorescent dye tagged cells in anannular biological fluid layer are now described.

With reference to FIG. 16, certain measurement parameters arediagrammatically illustrated. The objective 40 images over a field ofview (FOV) and over a depth of view located at a focus depth. In FIG.16, the focus depth is indicated respective to the objective 40;however, the focus depth can be denoted respective to another reference.In some embodiments, the depth of view of the objective 40 is about 20microns, while the annular gap 12 between the test tube wall 14 and thefloat wall 16 is about 50 microns. However, the depth of focuscorresponding to the annular gap 12 can vary substantially due tonon-uniformities in the test tube and/or the float or other factors. Itis expected that the annular gap 12 is located somewhere within anencompassing depth range. In some embodiments, an encompassing depthrange of 300 microns has been found to be suitable. These dimensions areexamples, and may be substantially different for specifice embodimentsdepending upon the specific objective 40, light-transmissive test tube,float, the type of centrifuging or other sample processing applied, andso forth.

With reference to FIG. 17, one suitable data acquisition approach 300 isdiagrammatically shown. In process operation 302, analysis images areacquired at a plurality of focus depths spanning the encompassing depthrange. To avoid gaps in the depth direction, the number of analysisimages acquired in the operation 302 should correspond to at least theencompassing depth range divided by the depth of view of the objective40.

In some embodiments, the analysis images are processed in optionaloperation 304 to identify one or more analysis images at about the depthof the biological fluid layer (such as the buffy layer) based on imagebrightness. This optional selection takes advantage of the observationthat typically the fluorescent dye produces a background fluorescencethat is detected in the acquired analysis images as an increased overallimage brightness. Image brightness can be estimated in various ways,such as an average pixel intensity, a root-mean-square pixel intensity,or so forth.

In an image processing operation 306, the analysis images, or those oneor more analysis images selected in the optional selection operation304, are processed using suitable techniques such as filtering,thresholding, or so forth, to identify observed features as candidatecells. The density of dye-tagged cells in the biological fluid layer istypically less than about one dye-tagged cell per field of view.Accordingly, the rate of identified candidate cells is typically low.When a candidate cell is identified by the image processing 306, asuitable candidate cell tag is added to a set of candidate cell tags310. For example, a candidate cell tag may identify the image based on asuitable indexing system and x- and y-coordinates of the candidate cellfeature. Although the density of rare cells is typically low, it iscontemplated that the image processing 306 may nonetheless on occasionidentify two or more candidate cells in a single analysis image. On theother hand, in some analysis images, no candidate cells may beidentified.

At a decision point 312, it is determined whether the sample scan iscomplete. If not, then the field of view is moved in operation 314. Forexample, the field of view can be relatively scanned across thebiological fluid sample in the annular gap 12 by a combination ofrotation of the test tube 72 and translation of the objective 40 alongthe test tube axis 75. Alternatively, using the tube holder of FIG. 13,scanning is performed by moving the test tube 72 spirally. For each newfield of view, the process operations 302, 304, 306 are repeated.

Once the decision point 312 indicates that the sample scan is complete,a user verification process 320 is optionally employed to enable a humananalyst to confirm or reject each cell candidacy. If the imageprocessing 306 is sufficiently accurate, the user verification process320 is optionally omitted.

A statistical analysis 322 is performed to calculate suitable statisticsof the cells confirmed by the human analyst. For example, if the volumeor mass of the biological fluid sample is known, then a density of rarecells per unit volume or per unit weight (e.g., cells/milliliter orcells/gram) can be computed. In another statistical analysis approach,the number of confirmed cells is totaled. This is a suitable metric whena standard buffy sample configuration is employed, such as a standardtest tube, standard float, standard whole blood sample quantity, andstandardized centrifuging processing. The statistical analysis 322 mayalso include threshold alarming. For example, if the cell number ordensity metric is greater than a first threshold, this may indicate aheightened possibility of cancer calling for further clinicalinvestigation, while if the cell number or density exceeds a second,higher threshold this may indicate a high probability of the cancercalling for immediate remedial medical attention.

With reference to FIG. 18, a modified acquisition approach 300′ isdiagrammatically shown. In modified process operation 304′, the focusdepth for maximum background fluorescence intensity is first determinedusing input other than analysis images, followed by acquisition 302′ ofone or a few analysis images at about the focus depth for maximumbackground fluorescence. For example, the search process 304′ can beperformed by acquiring low resolution images at various depths. To avoidgaps in the depth direction, the number of low resolution imagesacquired in the operation 304′ should correspond to at least theencompassing depth range divided by the depth of view of the objective40. In another approach, a large-area brightness sensor (not shown) maybe coupled to the captured fluorescence 50 (for example, using a partialmirror in the camera 56, or using an intensity meter built into thecamera 56) and the focus of the objective 40 swept across theencompassing depth range. The peak signal of the sensor or meter duringthe sweep indicates the focus providing highest brightness.

With the depth of the biological fluid sample determined by the processoperation 304′, the acquisition process 302′ acquires only one or a fewanalysis images at about the identified focus depth of highestbrightness. To ensure full coverage of the biological fluid layer, thenumber of acquired analysis images should be at least the thickness ofthe annular gap 12 divided by the depth of view of the objective 40. Forexample, if the annular gap 12 has a thickness of about 50 microns andthe depth of view is about 20 microns, then three analysis images aresuitably acquired—one at the focus depth of highest brightness, one at afocus depth that is larger by about 15-25 microns, and one at a focusdepth that is smaller by about 15-25 microns.

An advantage of the modified acquisition approach 300′ is that thenumber of acquired high resolution analysis images is reduced, since thefocus depth is determined prior to acquiring the analysis images. It isadvantageous to bracket the determined focus depth by acquiring analysisimages at the determined focus depth s and at slightly larger andslightly smaller focus depths. This approach accounts for thepossibility that the rare cell may be best imaged at a depth thatdeviates from the depth at which the luminescence background is largest.

With reference to FIG. 19, a suitable embodiment of the image processing306 is described, which takes advantage of a priori knowledge of theexpected rare cell size to identify any cell candidates in an analysisimage 330. In a matched filtering process 332, a suitable filter kernelis convolved with the image. The matched filtering 332 employs a filterkernel having a size comparable with the expected size of an image of arare cell in the analysis image 330.

With continuing reference to FIG. 19 and with brief further reference toFIGS. 20 and 21, in some embodiments a square filter kernel 334 isemployed. The kernel 334 includes a central positive region of pixelseach having a value of +1, and an outer negative region of pixels eachhaving a value of −1. The area of the positive region should be aboutthe same size as the area of the negative region. Points outside ofeither the inner or outer region have pixel values of zero. Optionally,other pixel values besides +1 and −1 can be used for the inner and outerregions, respectively, so as to give the filter a slightly positive orslightly negative response.

With continuing reference to FIG. 19, the matched filtering removes orreduces offsets caused by background illumination, and also improves thesignal-to-noise ratio (SNR) for rare cells. The signal is increased bythe number points in the positive match area, while the noise isincreased by the number of points in both the positive and negativematch areas. The gain in SNR comes from the fact that the signaldirectly adds, while the noise adds as the root-mean-square (RMS) valueor as the square root of the number of samples combined. For a filterwith N positive points and N negative points, a gain of N/√(2N) or√(N/2) is obtained.

The square filter kernel 334 is computationally advantageous since itsedges align with the x- and y-coordinate directions of the analysisimage 330. A round filter kernel 334′ or otherwise-shaped kernel isoptionally used in place of the square filter kernel 334. However, theround filter kernel 334′ is more computationally expensive than thesquare filter kernel 334. Another advantage of the square filter kernel334 compared with the round filter kernel 334′ is that the total filteredge length of the square filter 334 is reduced from twice the detectionsize to 1.414 times the detection size. This reduces edge effects,allowing use of data that is closer to the edge of the analysis image330.

The size of the filter kernel should be selected to substantially matchthe expected image size of a dye-tagged cell in the analysis image 330to provide the best SNR improvement. For example, the square filterkernel 334 with a positive (+1) region that is ten pixels across isexpected to provide the best SNR improvement for a cell image alsohaving a diameter of about ten pixels. For that matched case, the signalis expected to increase by about a factor of 78 while the noise isexpected to increase by about a factor of 14, providing a SNRimprovement of about 5.57:1. On the other hand, the SNR improvement fora smaller eight pixel diameter cell using the same square filter isexpected to be about 3.59:1. The SNR improvement for a larger fourteenpixel diameter cell using the same square filter is expected to be about3.29:1.

The matched filter processing 332 can be implemented in various ways. Inone approach, each point in the input image is summed into all points inthe output image that are in the positive inner region. Then all thepoints in the output image that are in the outer negative region but notin the inner positive region are subtracted off. Each point in the inputimage is touched once, while each point in the output image is touchedthe outer-box pixel area count number of times.

In another suitable approach, for each point in the output image, allpoints from the input image that are within the positive inner box areread and summed. All points outside the positive inner box but withinthe negative outer box are then subtracted. While each output imagepixel is touched only once, each input image pixel is touched by theouter-box pixel count.

In another suitable approach, two internal values are developed for thecurrent row of the input image: a sum of all points in the row in thenegative outer box distance, and a sum of all points in the row in theinner positive box distance. All output image column points at thecurrent row have the input image sum of all points in the outer-boxsubtracted from them. All the output image column points within theinner positive box get the sum of the input image row points in theinner positive box distance added in twice. The row sums can be updatedfor the next point in the row by one add and one subtract. This reducesthe execution cost to be on the order of the height of the filter box.

In the matched filter processing 332, various edge conditions can beemployed. For example, in one approach, no output is produced for anypoint whose filter overlaps an edge of the analysis image 330. Thisapproach avoids edge artifacts, but produces an output image of reducedusable area. In another suitable example edge condition, a default value(such as zero, or a computed mean level) is used for all points off theedge.

With continuing reference to FIG. 19, binary thresholding processing 338is applied after the matched filtering 332. A difficulty in performingthe thresholding 338 is selection of a suitable threshold value.Threshold selection is complicated by a likelihood that some analysisimages will contain no cells, or only a single cell, or only a couple orfew cells. In one approach, a the threshold is selected as a value thatis a selected percentage below the peak pixel intensity seen in thefiltered data. However, this threshold will cause noise to be detectedwhen no cells are present, since in that case the peak pixel value willbe in the noise. Another approach is to use a fixed threshold. However,a fixed threshold may be far from optimal if the background intensityvaries substantially between analysis images, or if the matchedfiltering substantially changes the dynamic range of the pixelintensities.

In the illustrated approach, the threshold is determined by processing340 based on the SNR of the unfiltered analysis image 330. By firstdetermining the standard deviation of the input image, the expectednoise at the filter output can be computed. The noise typically rises bythe square root of the number of pixels summed, which is the outer-boxarea in pixel counts. In some embodiments, the threshold is set atapproximately 7-sigma of this noise level. As this filter does not havean exact zero DC response, an appropriate mean level is also suitablysummed to the threshold.

The thresholding 338 produces a binary image in which pixels that arepart of a cell image generally have a first binary value (e.g., “1”)while pixels that are not part of a cell image generally have secondbinary value (e.g., “0”). Accordingly, connectivity processing 344 isperformed to identify a connected group of pixels of the first binaryvalue corresponding to a cell. The connectivity analysis 344 aggregatesor associates all first binary value pixels of a connected group as acell candidate to be examined as a unit. The center of this connectedgroup or unit can be determined and used as the cell locationcoordinates in the candidate cell tag.

With reference to FIG. 22, a suitable embodiment of the optional userverification processing 320 is described. A tag is selected forverification in a selection operation 350. In a display operation 352,the area of the analysis image containing the candidate cell tag isdisplayed, optionally along with the corresponding area of analysisimages adjacent in depth to the analysis image containing the candidatecell. Displaying the analysis images that are adjacent in depth providesthe reviewing human analyst with additional views which may fortuitouslyinclude a more recognizable cell image than the analysis image in whichthe automated processing 306 detected the cell candidate. The humananalyst either confirms or rejects the candidacy in operation 354. Aloop operation 356 works though all the candidate cell tags to providereview by the human analyst of each candidate cell. The statisticalanalysis 322 operates on those cell candidate tags that were confirmedby the human analyst.

Example data acquisition and analysis processing has been described withreference to FIGS. 16-22 in the context of quantitative buffy coatanalysis using the annular sample in the annular gap 12 between the testtube wall 14 and the float wall 16. However, it will be appreciated thatthe processing is readily applied to other sample scanning approaches,such as the scanning of the planar sample slide 60 depicted in FIG. 4.

The example embodiments principally relate to quantitative buffy coatanalysis. However, it will be appreciated that the apparatuses andmethods disclosed herein are applicable to other types of bioassays. Forexample, the cells can be stained rather than fluorescently tagged, orthe cells may have an intrinsic optical signature (fluorescence,contrast, or so forth) that enables assessment by optical microscopy.The features assessed may be other than rare cells. For example, theassessed features may be cell fragments, bacteria, or multi-cellularstructures. The sample may be a biological sample other than a buffycoat sample.

The invention has been described with reference to the preferredembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such modifications and alterations insofar as they come within thescope of the appended claims or the equivalents thereof.

1. A method for identifying fluorescent dye-tagged rare cells in abiological fluid layer contained in an annular gap between an insidetest tube wall and a float wall, the method including: acquiring one ormore analysis images at one or more focus depths using a microscopesystem; image processing each acquired analysis image to identifycandidate rare cell images; selectively rotating the test tube;selectively relatively translating the test tube and the microscopesystem along a test tube axis; and repeating the acquiring and imageprocessing for a plurality of fields of view accessed by the selectiverotating and selective relative translating and substantially spanningthe annular gap.
 2. The method as set forth in claim 1, furthercomprising: displaying each identified candidate rare cell image to anassociated human analyst; and receiving from the associated humananalyst an indication of whether the displayed candidate rare cell imagecorresponds to a dye-tagged rare cell.
 3. The method as set forth inclaim 2, wherein the displaying includes: displaying an image areacontaining the candidate rare cell image extracted from the analysisimage containing the candidate rare cell image; and displaying at leastone corresponding image area extracted from another analysis image thatis adjacent in depth to the analysis image containing the candidate rarecell image.
 4. The method as set forth in claim 1, further comprising:determining the one or more focus depths for the acquiring of the one ormore analysis images based on a background fluorescence of thebiological fluid layer.
 5. An apparatus comprising a non-volatilestorage medium encoding instructions executable by a digital processorto perform a method for identifying fluorescent dye-tagged cells inmicroscope-acquired analysis images of a biological fluid layer having adensity of the dye-tagged cells less than about one dye-tagged cell peranalysis image, the method comprising: image processing each analysisimage to identify candidate features having a feature size substantiallycorresponding to an expected size of fluorescent dye-tagged cell imagesin the analysis images.
 6. The apparatus as set forth in claim 5,wherein the image processing comprises: matched-filtering each analysisimage using a filter kernel having a size substantially corresponding tothe expected size of fluorescent dye-tagged cell images in the analysisimages.
 7. The apparatus as set forth in claim 6, wherein the filterkernel is square.
 8. The apparatus as set forth in claim 6, wherein thefilter kernel is circular.
 9. The apparatus as set forth in claim 6,wherein the image processing of each analysis image further comprises:thresholding the matched-filtered analysis image to generate acorresponding binary image; and identifying the candidate features basedon connectivity analysis of the binary image.
 10. The apparatus as setforth in claim 9, wherein the thresholding employs a threshold computedfrom a signal-to-noise ratio of the analysis image.
 11. A method foridentifying fluorescent dye-tagged cells in a biological fluid layerusing a microscope system having a field of view at a selectable focusdepth, the microscope field of view being substantially smaller than anarea of the biological fluid layer, the method comprising: identifying adepth of the biological fluid layer based on a background fluorescenceof the biological fluid layer caused by the fluorescent dye-tagging;acquiring one or more analysis images using the microscope system at oneor more focus depths at or close to the identified depth of thebiological fluid layer; image processing the acquired one or moreanalysis images to identify candidate features that may correspond todye-tagged cells; and relatively moving the field of view and thebiological fluid layer; and repeating the identifying, acquiring, imageprocessing, and relative moving to identify candidate features over thearea of the biological fluid layer.
 12. The method as set forth in claim11, wherein the identifying comprises: acquiring a plurality ofdepth-sampling images at different focus depths spanning a pre-selectedencompassing depth range; and identifying the depth of the biologicalfluid layer as corresponding to the focus depth of the brightestacquired depth-sampling image.
 13. The method as set forth in claim 12,wherein the one or more analysis images include at least the brightestacquired depth-sampling image.
 14. The method as set forth in claim 12,wherein the one or more analysis images include at least the brightestdepth-sampling image and two additional depth-sampling images havingfocus depths closest to the optical focus depth of the brightestdepth-sampling image.
 15. The method as set forth in claim 12, whereinthe one or more analysis images are acquired at a higher resolution thanthe depth-sampling images and are acquired after the acquiring of theplurality of depth-sampling images.
 16. The method as set forth in claim12, wherein the number of depth-sampling images is greater than or equalto the pre-selected encompassing depth range divided by a depth-of-viewof the microscope system.
 17. The method as set forth in claim 11,wherein the identifying of the depth of the biological fluid layercomprises: illuminating the biological fluid layer through an objectiveof the microscope system; and identifying the focus depth of theobjective of the microscope system at which the illuminating produces ahighest background fluorescence observed through the objective.
 18. Themethod as set forth in claim 11, wherein a thickness of the biologicalfluid layer is greater than a depth-of-view of the microscope system,and the acquiring of the one or more analysis images comprises:acquiring a number of analysis images greater than or equal to thethickness of the biological fluid sample divided by the depth-of-view ofthe fluorescent imager.
 19. The method as set forth in claim 11, whereinthe image processing of each analysis image comprises: matched-filteringthe analysis image using a filter kernel sized to substantially matchimages of fluorescent dye-tagged cells in the analysis images.
 20. Themethod as set forth in claim 19, wherein the image processing of eachanalysis image further comprises: thresholding the matched-filteredanalysis image using a threshold computed from a signal-to-noise ratioof the analysis image to generate a corresponding binary image in whichthe candidate features are identified.
 21. The method as set forth inclaim 11, further comprising: disposing a biological fluid samplecontaining the fluorescent dye-tagged cells in a test tube; disposing afloat in the test tube, the float defining an annular gap between anouter surface of the float and an inner surface of the test tube; andcentrifuging the test tube with the biological fluid sample and thefloat disposed therein, the centrifuging causing a component of thebiological fluid sample including the fluorescent dye-tagged cells tosegregate at the annular gap to define the biological fluid layer. 22.The method as set forth in claim 21, wherein the biological fluid sampleis a blood sample, and the segregated biological fluid layer is a buffycoat containing fluorescent dye-tagged epithelial cells.
 23. The methodas set forth in claim 21, wherein the float includes at least twoannular ridges engaging with the inner surface of the test tube to sealthe annular gap, the annular ridges disengaging from the inner surfaceof the test tube during centrifuging to admit the segregating componentof the biological fluid sample into the annular gap.